Insitu assay of substance in biological sample using labeled probe

ABSTRACT

A method for analyzing an objective substance, comprising reacting a labeled probe with an objective substance on a biological sample, said probe comprising a label substance of the formula (I):                    
     wherein A 1  is an aromatic group, R 1  is a hydrogen or —COCH 2 COC n F 2n+1  and n is an integer of 1-6, which is bonded to a probe selected from the group consisting of nucleic acid, nucleic acid binding protein, low molecular ligand and receptor for ligand (except antibody) to give a fluorescent complex, reacting the complex with an objective substance on a biological sample and assaying fluorescence of the resultant fluorescent complex, a labeled nucleic acid probe and a labeled nucleotide. According to the method of the present invention, defects such as hindrance of fluorescence due to contaminant substance, low sensitivity and the like can be resolved, thereby enabling analysis on a tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 09/301,406, filed Apr. 28, 1999, now U.S. Pat No. 6,339,172.

FIELD OF THE INVENTION

The present invention relates to a novel in situ assay method for anobjective substance in a biological sample, comprising assaying on saidbiological sample, a reagent therefor, particularly, a novel labelednucleic acid probe and a fluorescent complex comprising said probe and aheavy metal ion, a labeled nucleotide for preparing said labeled nucleicacid probe and a process for preparing said labeled nucleic acid probe.More particularly, the present invention relates to a novel method whichcan be preferably used for analyzing the function and behavior of acertain substance (e.g., nucleic acid) on a biological sample (e.g., abiological tissue and a cell), by assaying the localization orconcentration thereof on the biological sample, as well as a labeledprobe and a reagent for analysis which contains said probe to be usedfor said method.

BACKGROUND OF THE INVENTION

In the research field of life science and the field of clinicaldiagnostic and clinical tests, fluorescent substances have been widelyused as a label substance, besides radioactive substances, enzymes andthe like. With the progress of the image analyzing technique systems inrecent years, they have been more increasingly used in a broad range ofapplications, thereby providing new findings in the function andbehavior of biological substances in a living body.

Such fluorescent substances typically include compounds comprisingfluorescein, dansyl group, anthraniloyl group, pyrene, rhodamine,nitrobenzoxadiazl and the like.

The fluorescent substances, which are intercalated in between doublestrands of nucleic acid (DNA) and enable fluorescent staining of theDNA, include Hoechst 33342 manufactured by Molecular Probe, 4′,6′-diamino-2-phenylindol dihydrochloride (DAPI), propidium iodite (PI),acridium orange and the like. Besides these, commercially availableproducts such as SYTO (TM), BOBO (TM), POPO (TM), TOTO (TM), YOYO (TM)and the like are used similarly.

To label lipids, fluorescent substances, such as4-nitrobenzene-2-oxa-1,3-diazol (NBD) and4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), are used.

In recent years, fluorescent substances capable of labeling various ionsor other low molecular substances (e.g., fura-2, indo-1, fluo-3, etc.for calcium ion, SBFI, etc. for sodium ion, mag-fura-2, mag-indo-1, etc.for magnesium ion, TSQ, etc. for zinc ion, SPQ, etc. for chloride ionand FICRhR for cyclic AMP) have been developed, and the behavior of ionsin a living body has been studied using these fluorescent substances.

In an assay of a substance in a biological sample, it is desired of afluorescent substance, theoretically and practically, that (1) it doesnot deactivate nucleic acid, peptide, low molecular ligand and the likeafter binding, (2) it has a high fluorescence quantum yield and highphotostability, (3) its fluorescence lifetime is long, (4) it is free ofthe effect of other endogenous fluorescent substances in the biologicalsample, (5) it does not react non-specifically with an endogenousmolecule in the biological sample, (6) it easily dissolves in water and(7) its determination is convenient. Particularly, in an in situ assayon a tissue or cell, a fluorescent substance is further required to notreact non-specifically with a biomolecule present in the tissue or cellor on the surface thereof.

However, some of the above-mentioned fluorescent substances are unstableto light and/or heat, some have low quantum yield, and others have shortexcited fluorescent lifetime and are subject to the effect ofautofluorescence of other endogenous substances. The conventionallyknown fluorescent substances are not ideal fluorescent compounds, butrather, are insufficient, since they are more or less problematic in oneor more aspects, such as low S/N ratio, short fluorescence wavelengthand the like.

The influence of endogenous fluorescence in the assay of substances in aliquid sample such as body fluid and cell extract can be removed byusing, as a lanthanoide metal-containing fluorescent complex, a complexlabeled with a novel fluorescent substance and consisting of a substancehaving affinity for the objective substance and an europium ion. Amethod has been developed which is free of an influence of thebackground fluorescence derived from a fluorescent substance ornon-fluorescent substance in a biological sample, particularly a serum,during assay of a physiologically active substance in the sample, andwhich comprises subjecting the complex to a time-resolved fluorescenceassay.

For example, use of a diazophenyl-EDTA-europium complex orisothiocyanatephenyl-EDTA-europium complex for an immunoassay has beenknown (Anal Biochem, 137, 335-343, 1984). In this immunoassay,β-naphthoyltrifluoroacetone (β-NTA) is added to the assay system in theco-existence of β-diketone and tri-n-octyl-phosphine oxide (TOPO) toachieve the highest sensitivity.

This assay system has been known as a DELFIA system (DissociationEnhanced Lanthanide Fluoroimunmunoassay). A method utilizing a europiumcomplex represented by this system is advantageous in that an assaytarget in a biological sample can be detected without the influence offluorescence having a short lifetime which is derived from acontaminating substance in the body, due to the fluorescent property ofthis complex that its life is long.

On the other hand, the DELFIA system is associated with the defectcaused by a reaction between β-NTA or TOPO used for the assay andeuropium in a sample or in the environment, thereby producing strongfluorescence, which may prevent detection of the assay target.

In addition to the inherent defect this system has in that it issusceptible to the influence of the contaminated europium, the need toadd a fluorescence intensifier such as β-NTA makes the assay on a solidphase unattainable. There is also a problem of manipulative complexitydue to the step of adding a fluorescence intensifier. In conclusion, insitu assay of a physiologically active substance (e.g., nucleic acid,receptor, sugar chain, ganglioside and the like) fixed on a tissue orcell (surface) by this system is extremely difficult.

To resolve the above-mentioned defects of the DELFIA system, a CyberFluor system that uses a complex of4,7-bis-(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid(BCPDA) and europium is known (Anal. Chem, 61, 48-53, 1989).

The use of BCPDA has made a great advancement in that many europiumfluorescent complexes can be introduced without a quenching phenomenon(quenching phenomenon strikingly decreases the fluorescence quantumyield) caused when one probe is, labeled with many fluoresceins and thatit is highly stable and can resolve the defects of the DELFIA system.

However, the Cyber Fluor system has a fatal defect in that itssensitivity is lower than that of the DELFIA system by the order of twodigits or more. To compensate for the defect, synthesis of a number ofeuropium complexes was tried, and, for example, trisbipyridine cryptate(TBP) europium complex and the like are known (Clin. Chem, 196-201,1993, U.S. Pat. No. 5,262,526, JP 07-10819 A and the like). These newlydeveloped europium fluorescent complexes have defects in that they haveshort excitation wavelengths and weak fluorescence, and they requiremany synthetic steps. Thus, they do not have particularly superiorproperty as compared to the above-mentioned two europium fluorescentcomplexes.

Many studies have been made so far with respect to europium fluorescentcomplex and it has been found that β-diketone-europium fluorescentcomplex has greater fluorescence intensity than aromatic amine-europiumcomplex, and of the β-diketone ligands, a europium fluorescent complexof 2-naphthoyltrifluoroacetone (β-NTA) and 2-thenoyltrifluoroacetone(TTA) particurlaly has the greatest fluorescence intensity.

The present inventors synthesized various β-diketonato-europium TOPOcomplexes to study the effect of β-diketone as a substituent on thefluorescence property of the β-diketone-europium fluorescent complex,and found that the fluorescence intensity of these complexes isdependent on the composition and structure of the substituents R¹ and R²of the β-diketonato (R¹COCH₂COR²). In other words, when R¹ is anaromatic hydrocarbon residue, stronger electron attractiveness of R²results in stronger fluorescence intensity of the complex, based onwhich finding an immunoassay utilizing a β-diketone type europiumfluorescent complex having a dramatically improved fluorescenceintensity has been found (U.S. Pat. No. 5,859,297 and Anal. Chem, 70,596-601, 1988).

However, the use of this β-diketone type europium fluorescent complexfor the assay of a substance having various actions that is on abioloical tissue or cell, such as a physiologically active substance,has not been disclosed. Many difficulties are foreseeable in an assay ona tissue or cell of a physiologically active substance in the biologicalsample, for example, a great influence of contaminating substance, adifficult high sensitivity assay, an unattainable easy assay and thelike.

It is therefore an object of the present invention to provide a means ofresolving defects such as hindrance of fluorescence by a contaminatingsubstance and low sensitivity, so that a substance in a tissue or cellor on surface thereof, such as nucleic acid, nucleic acid bindingprotein, receptor, sugar chain, ganglioside and the like can be assayedas it is on the tissue or cell with high precision and high sensitivity.

SUMMARY OF THE INVENTION

The present invention is based on the finding that a β-diketone formeuropium fluorescent complex has superior characteristics as a label forprobe for the high sensitivity assay of a physiologically activesubstance such as nucleic acid, nucleic acid binding protein, receptor,enzyme, sugar chain, ganglioside and the like on a tissue or cell, sinceit has a noticeably long fluorescence lifetime and permits time-resolvedfluorescence assay, assay upon elimination of blank fluorescence, use inone step: and has a long wavelength fluorescence lifetime.

Accordingly, the present invention provides a method for analyzing abiological substance comprising the use of a label substance of thefollowing formula (I):

wherein A¹ is an aromatic group, R¹ is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, or a label substanceof the following formula (II)

wherein A² and A³ are the same or different and each is an aromaticgroup, R² and R³ are the same or different and each is a hydrogen orCOCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, reagents therefor and apreparation method thereof. More particularly, the present inventionprovides the following.

(1) A method for analyzing an objective substance, comprising reacting alabeled probe with an objective substance on a biological sample, saidprobe comprising a label substance of the formula (I) or a labelsubstance of the formula (II) bonded to a probe selected from the groupconsisting of nucleic acid, nucleic acid binding protein, low molecularligand and receptor for ligand (except antibody) via a cross-linkinggroup or a cross-linking group and a conjugating group, adding a heavymetal ion and assaying fluorescence of the resultant fluorescentcomplex.

(2) The method for analyzing an objective substance, comprising adding aheavy metal ion to a labeled probe, said probe comprising a labelsubstance of the formula (I) or a label substance of the formula (II)bonded to a probe selected from the group consisting of nucleic acid,nucleic acid binding protein, low molecular ligand and receptor forligand (except antibody) via a cross-linking group or a cross-linkinggroup and a conjugating group to give a fluorescent complex, reactingthe complex with an objective substance on a biological sample andassaying fluorescence of the resultant fluorescent complex.

(3) A labeled nucleic acid probe comprising a label substance of theformula (I) or a label substance of the formula (II) bonded to a nucleicacid probe via a cross-licking group.

(4) A fluorescent complex comprising the labeled nucleic acid probe of(3) and a heavy metal ion.

(5) A reagent for analyzing a nucleic acid, comprising the labelednucleic acid probe of (3).

(6) A labeled nucleotide comprising a label substance of the formula (I)bonded to a nucleotide via a cross-linking group.

(7) A fluorescent complex comprising the labeled nucleotide of (6) and aheavy metal ion.

(8) A method for producing a labeled nucleic acid probe comprisingreacting the labeled nucleotide of (6), dNTPs and a single strand DNA inthe presence of a DNA polymerase.

(9) A method for producing a labeled nucleic acid probe comprisingreacting the labeled nucleotide of (6), dNTPs and a double stranded DNAin the presence of 5′-exonuclease, DNase and a DNA polymerase.

(10) A labeled nucleic acid probe obtained by the production method of(8) or (9).

(11) A reagent for analyzing nucleic acid comprising a label substanceof the formula (I) or the formula (II), to which avidin is covalentlybonded via a cross-linking group (hereinafter to be referred to as labelsubstance A) and a nucleotide to which biotin is bonded via a linkagegroup (hereinafter to be referred to as nucleotide B).

(12) A method for analyzing an objective substance comprising reactingthe objective substance with a nucleic acid probe comprising thenucleotide B as a component on a biological sample and then with thelabel substance A, adding a heavy metal ion and assaying thefluorescence of the resultant fluorescent complex.

According to the method of the present invention, defects such ashindrance of fluorescence by a contaminating substance and lowsensitivity can be resolved in the analysis of nucleic acid, nucleicacid binding protein, receptor, sugar chain, ganglioside and the like ona biological tissue, cell or chromosome in a biological sample. Inparticular, hindrance due to contamination with a lanthanoide metal ionin a sample or environment can be removed, and assay of the objectivesubstance with high sensitivity and with small steps.

DETAILED DESCRIPTION OF THE INVENTION

The label substance in the present invention is represented by theformula (I) or the formula (II). In the formulas, A¹, A² and A³ are thesame or different and each is a trivalent aromatic group, particularly aconjugated double bond, wherein when R¹, R² or R³ is hydrogen, A¹, A² orA³ it binds with is a divalent aromatic group. Such divalent ortrivalent aromatic group is exemplified by

and the like. Those having a substituent to these aromatic rings, suchas methylphenylene and methyldibenzothiopehne, are also exemplified.

Particularly preferable aromatic group is the following:

R¹, R² and R³ are each independently hydrogen or COCH₂COC_(n)F_(2n+1).

In the formulas (I), (II) and (III), n and n at R¹, R² and R³ are aninteger of 1-6, preferably 2-4.

In the present invention, a particularly preferable label substance isrepresented by the formula (III):

wherein n is an integer of 1-6.

In the present invention, the probe is selected from the groupconsisting of nucleic acid, nucleic acid binding protein, ligand andreceptor for ligand (except antibody).

In the present invention, the objective substance is a component in thebiological sample, which is to be the subject of the analysis.Preferable examples thereof include nucleic acid, nucleic acid bindingprotein, ligand, receptor for ligand and the like.

The nucleic acid binding protein is a protein that specifically bindswith the nucleic acid having a specific nucleotide sequence, such ashistone, DNA binding protein, Lac I protein and the like. By the use ofa transcription regulating factor of cytokine (a kind of DNA bindingproteins), such as NF-κB and the like, as a probe to be labeled, theinteraction between the transcription factor and DNA can be visualized.

The low molecular ligand here means an organic compound such as sugarchain, aromatic compound, ganglioside, oligosaccharide, peptideconsisting of 2-10 amino acids and the like. Examples thereof includemyc peptide, thyroxine, triiodothyronine, ganglioside GM2, cellobiose,sugar chain having a sialic acid at the end thereof and the like.

The receptor for ligand means a substance that specifically binds with aspecific ligand that is located on or in a cell or between cells, suchas cellulose binding protein, sialic acid binding lectin, albuminreceptor and the like.

Further examples of the low molecular ligand or receptor include hormoneor hormone receptor such as insulin, insulin receptor, EGF, EGFreceptor, HGF, HGF receptor, TSH, TSH receptor and the like, andreceptors of low molecular ligands such as receptor of cytokine (e.g.,IL-8 and the like) or chemokine, acetylcholine receptor, histaminereceptor and the like.

The protein kinase C can bind with the derivative of phorbol ester andcan be assayed by the method of the present invention. In addition, theenzymes such as cAMP-dependent protein kinase, cGMP-dependent proteinkinase, calmodulin-dependent phosphoenzme, trosine-phosphorylated enzymeand the like can be assayed by way of ligand-receptor reaction, whereinthe labeled probe of the present invention can be used as the probe forthe substrate binding site of the enzyme.

Various lectins against various sugar chain and ganglioside can be usedas the probe of the present invention. Examples of lectin includeconcanavalin A against D-mannose bonded with various proteins on thecell, wheat germ agglutinin against di-N-acetylchitobiose, sialic acidbinding lectin against sialic acid, which is derived from Limuluspolyphemus and the like.

Examples of nucleic acid and nucleic acid probe include DNAs having aseries of various deoxyribonucleic acids (dATP, dGTP, dTTP, dCTP, dUTP)and RNAs having a series of various ribonucleic acids (rATP, rGTP, rTTP,rCTP, rUTP). These nucleic acid probe has a nucleotide sequence consistsof cDNA or antisense oligonucleotides that specifically hybridizes withmRNA which expresses in the cell. Alternatively, a nucleic acid probehaving a nucleotide sequence complementary to a part of a specificsequence of nucleic acid or chromosome in the cell can be used.

Examples of the nucleic acid or gene on chromosome in the cell includeoncogene (e.g., abl, erb, fos, myb, myc, ras, src and the like), tumorsuppressor gene (e.g., p53 and the like), rearranged T cell receptorgene, rearranged irmunoglobulin gene, a part of the nucleotide sequenceof pathogenic virus gene such as Epstein-Bar virus (EBV), herpes simplexvirus (HSV), cytomegalovirus (CMV), hepatitis B virus (HBV), rotavirus,adenovirus and the like, a part of the nucleotide sequence of infectiouspathogenic microorganism gene such as malaria protozoa, fungus,mycoplasma and the like, and nucleic acid having a nucleotide sequencecomplementary thereto.

A part or the whole of the nucleic acid probe complementary to thesegenes or homologous therewith may have a modified group such as methylgroup and the like as long as it does not affect bonding with a labelsubstance.

The labeled nucleic acid probe of the present invention is a compoundhaving affinity for a specific substance particularly on the tissue orcell, such as nucleic acid, nucleic acid binding protein and the likecontaining the above-mentioned genes on chromosome and the like.

The labeled probe in the present invention consists of a probe selectedfrom the group consisting of nucleic acid, nucleic acid binding protein,low molecular ligand and receptor for ligand (except antibody) and alabel substance bonded thereto. The labeled nucleic acid probe of thepresent invention consists of a nucleic acid and a label substancebonded to each other. The labeled nucleotide of the present inventionconsists of a nucleotide and a label substance bonded thereto. The bondbetween the label substance and the probe or nucleotide is a bond via across-linking group. It may be a covalent bond via a conjugating group.

The cross-linking group is via a bond between a label substance and aconjugating group, probe, nucleotide or avidin. That is, in a labeledprobe and a labeled nucleotide having a conjugating group, theconjugating group exists between the cross-linking group and the probeor nucleotide.

The label substance A in the present invention consists of avidin and alabel substance bonded via a cross-linking group. The binding ratio ofavidin-label substance is 1-50, preferably 2-30. The nucleotide B in thepresent invention consists of biotin and nucleotide bonded via a linkagegroup.

Avidin in the present invention is a glycoprotein that is contained inthe egg white and specifically binds with biotin. Avidin may be astreptoavidin derived from a microorganism (genus Streptococts) or arecombinant protein thereof.

Biotin in the present invention is a substance called vitamin H andcoenzyme R and binds extremely firmly with avidin or streptoavidin,wherein the bonding strength is far greater than the bond of typicalimmunoconjugate.

The cross-linking group in the present invention is derived from a groupcapable of bonding with both nucleic acid, nucleic add binding protein,low molecular ligand, receptor for ligand, nucleotide or avidin andaromatic group. Alternatively, it is derived from a group capable ofbonding with both linkage group and aromatic group.

Examples of the cross-linking group include —NH—CS—, —NH—CO—, —CO—,—N₂—, —NH—, —SO₂—, —CH₂S—, —CH₂—NH—, —(CH₂)₆—NH—CO—CH₂—CH₂—CO—, and S—S—and the like. Particularly preferable cross-linking groups are sulfonylgroup and carbonyl group.

The linkage group in the present invention is free of particularlimitation as long as it connects a cross-linking group and a nucleicacid, nucleic acid binding protein, low molecular ligand, receptor forligand or nucleotide. Preferable linkage group is a divalent aliphatichydrocarbon group having 5-25 carbon atoms and 7 or less amide bondsbetween carbons. Specific examples include a group of the formula (IV):

—CH═CHCO—NH_(b)CH₂_(a)CO—NH _(b)CH₂_(a)CO—NH_(b)  (IV)

wherein a is an integer of 0-6 and b is 0 or 1.

Another preferable mode of the like group is a linkage group containingbiotin and avidin through affinity binding.

Biotin affinity binding with avidin is preferably further bonded to aprobe via a linkage group. Examples of preferable linkage group includedivalent aliphatic hydrocarbon group having 5 to 25 carbon atoms andoptionally having 7 or less amide bonds between carbons. Specifically,it is —CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂—, whereinpreferable bond is(probe)—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂—biotin:avidin—(cross-linkinggroup-label substance).

The linkage group binding biotin and nucleotide in nucleotide B is freeof limitation as long as it binds biotin and nucleotide. Preferablelinkage group include a divalent aliphatic hydrocarbon group having 5 to25 carbon atoms and optionally having 7 or less amide bonds betweencarbons. Specifically, it is—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂—, whereinpreferable bond is(nucleotide)—CH═CH—CO—NH—CH₂—CH₂—NH—(CO-CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂—(biotin).

When the label substance of the formula (II) is bonded to probe oravidin via two cross-linking groups, the two cross-linking groups may bethe same or different. Again, when it is bonded to probe or avidin viatwo conjugating groups, the two conjugating group may be the same ordifferent.

The label substance of the formula (II) can be used upon binding to twoprobes or avidin. The two probes may be the same or different two probesor avidin. By binding to two probes, a synergistic binding effect can beexpected.

For example, one example of the labeled nucleic acid probe of thepresent invention is a label substance bonded to different nucleic acidprobes. These probes are nucleic acid probes having nucleotide sequencescomplementary to or homologous with the same or different genes. Inother words, a probe having plural nucleic acids recognizing thespecific sites of the assay target binds with the nucleic acid in thecell having nucleotide sequences complementary hereto or nucleotidesequences homologous thereto and becomes a so-called divalent probe byforming a fluorescent complex upon addition of a heavy metal ion (e.g.,lanthanoide metal ion), thereby affording a possible synergistic effect.

Even when the two binding probes are the same, each can bind with anobjective substance having same plural specific sites. Thus, the effectis not a simple addition but expected to be synergistic.

In the analysis method of the present invention, different kinds oflabeled probes may be used simultaneously upon mixing.

While the binding ratio of the probe-label substance is free ofparticular limitation, it is generally 1-100, preferably 1-20.

The binding ratio of the nucleotide-label substance is free ofparticular limitation, it is generally 1-50, preferably 1-20.

The binding ratio of the avidin-label substance is free of particularlimitation, it is generally 1-50, preferably 2-30.

The labeled probe, labeled nucleotide or label substance A can beproduced by the use of the following functional groups as long as itdoes not exert an adverse influence, for binding a label substance tothe probe, nucleotide or avidin. For example, various binding groupssuch as isothiocyanate group reactive with amino group, sulfonyl halidegroup (sulfonyl chloride group, sulfonyl fluoride group and the like),o-phthalaldehyde group in the presence of 2-mercaptoethanol,N-substituted maleimide group and the like for carbodiimnide group andthiol group, iodoacetamide group for histidine and the like.

Specifically, a ligand, nucleic acid and the like and a labelingcompound of the following formula in a molar amount of 1-20 per mol ofligand, nucleic acid and the like are reacted in a solvent.

The present invention also relates to a fluorescent complex containing alabeled nucleic acid probe and a heavy metal ion. Examples of the heavymetal ion include lanthanoide metal ion and radium ion, with preferencegiven to lanthanoide metal ion. The lanthanoide metal ion to be used inthe present invention includes ions of europium (Eu), samarium (Sm),terbium (Tb), dysprosium (Dy) and the like. It is typically used in theform of a chloride, but may be used in the form of other salts as longas the assay is not influenced. In the present invention, theselanthanoide metal ions may be used alone or in combination.

The reaction between the labeled probe and the objective substance inthe present invention is the reactions between nucleic acid and nucleicacid, nucleic acid and nucleic acid binding protein, and ligand andreceptor for ligand in a biological sample. For facilitated reaction,the biological sample may be pre-treated. For example, nucleic acidextraction by AGPC, protein dissociation treatment with ethanol and thelike can be applied.

The biological sample is preferably a cell, tissue or chromosome.

The analysis method of the present invention analyzes the objectivesubstance in the cell and on the cell surface, wherein a labeled probeis reacted with the objective substance at a tissue section, on a cellsurface, on a chromosome and the like, a heavy metal ion such aslanthanoide metal ion, radium ion and the like is added and fluorescenceof the resultant complex is assayed, or a heavy metal ion such aslanthanoide metal ion, radium ion and the like is added to a labeledprobe to give a fluorescent complex, which is reacted with the objectivesubstance on a biological sample and fluorescence of the complex afterreaction is assayed.

The analysis method of the present invention may comprise reacting anucleic acid probe containing nucleotide B as a component with theobjective substance on a biological sample, then reacting with labelsubstance A, adding a heavy metal ion, and assaying fluorescent of theresultant fluorescent complex.

The nucleic acid probe containing nucleotide B as a component means thatone or more nucleotides in the nucleotide sequence is(are) nucleotide B.Namely, it is a nucleic acid probe binding with biotin.

The nucleic acid probe containing nucleotide B as a component can beobtained by reacting nucleotide B, dNTPs and single strand DNA in thepresence of a primer and a DNA polymerase to give a double stranded DNAand denaturing the obtained DNA with heat to give a single strand DNA.

The nucleic acid probe containing nucleotide B as a component can beobtained by reacting nucleotide B, dNTPs and a double stranded DNA inthe presence of 5′-exonuclease, DNase and a DNA polymerase to give adouble stranded DNA and denaturing the obtained DNA with heat to give asingle strand DNA.

More specific analysis method is exemplified by the method comprisingimmersing a biological sample in a buffer containing a labeled probe,incubating the sample to allow reaction of the objective substance andthe labeled probe, washing off excess labeled probe with the buffer,immersing the probe in a buffer containing lanthanoide metal ion to forma complex and assaying the fluorescence of the resultant complex.

In addition, a method is exemplified, which comprises admixing buffercontaining lanthanoide metal ion with a buffer containing a labeledprobe to form a complex, immersing a biological sample in this mixture,incubating the sample to allow reaction with the objective substance,washing off excess (labeled probe: lanthanoide metal ion) complex andassaying the fluorescence of the resultant complex on the biologicalsample.

As a different specific method, the following method is exemplified.That is, a double stranded DNA having, a sequence to be the assaytarget, nucleotide B and dNTPs are reacted in the presence of5′-exonuclease, DNase and a DNA polymerase to give a biotin-boundnucleic acid probe. A biological sample is immersed in a buffercontaining this biotin-bound nucleic acid probe and incubated to allowreaction of the objective substance and the biotin-bound nucleic acidprobe, and excess biotin-bound nucleic acid probe is washed off. Then,the sample is immersed in a buffer containing the label substance A tobind biotin and avidin, and excess label substance A is removed. Then,the sample is immersed in a buffer containing lanthanoide metal ion toform a complex and the fluorescence of the resultant complex is assayed.

By these methods, the presence of the objective substance in abiological sample such as a tissue, cell, chromosome and the like isvisualized and analyzed for localization and concentration. In addition,abnormalities with respect to the objective substance can be analyzed.

The visualized image obtained by the use of the inventive labeled probecan be retained through a fluorescence microscope, confocallaser-scanning microscope and the like. The fluorescence signal itselfis assayable with a fluorescence assay device, time-resolvedfluorescence assay device and the like.

In particular, the inventive labeled nucleic acid probe is reacted witha biological sample of a tissue, cell, chromosome and the like andvisualize the objective substance therein by colony hybridization,fluorescence in situ hybridization (FISH) of tissue and chromosome,nucleic acid sandwich hybridization, comparative genome hybridization(CGH) and the like.

The present invention also relates to a labeled nucleotide. Thenucleotide of the present invention itself has affinity with a specificsubstance on a tissue or cell. It may be used to produce a labelednucleic acid probe by binding with a different nucleotide or by nicktranslation method from a double stranded DNA

The nucleotide in the labeled nucleotide and nucleotide of nucleotide Bof the present invention is not particularly limited and is exemplifiedby ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, dTTP, dUTP and the like, withparticular preference given to dUTP.

The particularly preferable labeled nucleotide has the following formula(V):

wherein X is a conjugating group of the formula (IV) and Y is a sulfonylgroup or carbonyl group, R is a group of the formula

and p is 0 or 1.

The present invention further relates to a fluorescent complexcontaining the above-mentioned labeled nucleotide and a heavy metal ion.Examples of the heavy metal ion include the above-mentioned lanthanoidemetal ion and radium ion, with preference given to the above-mentionedlanthanoide metal ion.

A labeled probe can be obtained by incorporating a labeled nucleotidesuch as the labeled dUTP of the present invention and the like, whensynthesizing a fragmented probe DNA using DNA extracted from the tissueor cell, particularly chromosomal DNA. To be specific, labelednucleotide, dNTPs and double stranded DNA is reacted in the presence of5′-exonuclease, DNase and a DNA polymerase to give a labeled nucleicacid probe. Alternatively, a labeled nucleotide, dNTPs and a singlestrand DNA are reacted in the presence of a DNA polymerase to give alabeled nucleic acid probe. Particularly preferably, labeled nucleotideof the present invention, such as labeled dUTP and the like isincorporated by nick translation method to give a DNA or a DNA fragmentusable as a labeled nucleic acid probe.

Moreover, labeled nucleic acid probe, labeled nucleotide or nucleotide Bof the present invention can be incorporated into DNA or RNA by nucleicacid amplification by PCR (polymerase chain reaction) method, LCR(ligase chain reaction) method, NASBA method and the like. The obtainedDNA or RNA can be used for the analysis of the objective substance as alabeled nucleic acid probe or a biotin-bound nucleic acid probe.

The nucleic acid probe obtained by incorporating the labeled nucleotideor nucleotide B of the present invention, that comprises a DNAcomplementary to the DNA of a tissue or cell can be particularlysuitably used for the analysis of abnormalities in the chromosome of theobjective tissue or cell.

The reagent for the analysis of nucleic acid of the present inventioncontains the above-mentioned novel labeled nucleic acid probe or labelednucleotide. Preferably it contains a heavy metal ion such as theabove-mentioned lanthanoide metal ion, radium ion and the like.

The reagent for the analysis of nucleic acid of the present inventioncontains label substance A and nucleotide B. The reagent containinglabel substance A and nucleotide B preferably further contains dNTPs,primer, DNA polymerase and heavy metal. As a different mode, a reagentcontaining label substance A and nucleotide B preferably furthercontains dNTPs, 5′-exonuclease, DNase, DNA polymerase and heavy metal.

The present invention is explained in detail by illustrative referenceexamples and examples, to which the present invention is not limited inany way.

Reference Example 1 Synthesis of 4,4′-diacetyl-o-terphenyl

To a solution of CH₂Cl₂ (200 ml), AlCl₃ (210 mmol) and CH₃COCl (205mmol) was gradually added a solution of CH₂Cl₂ (100 ml) and o-terphenyl(100 mmol) dropwise with stirring at 0° C. The mixture was stirred at 0°C. for 30 min and further stirred at room temperature for 24 hr. Thereaction solution was refluxed for 2 hr, and then poured into conc.hydrochloric acid with ice. The mixture was sufficiently stirred, andCH₂Cl₂ was distilled away under reduced pressure. The precipitate wasseparated by filtration, and thoroughly washed with water. The productwas recrystallized from 2-butanone (about 250 ml) to give needlecrystals, which were separated by filtration and dried in vacuo. Theyield was 22.1 g (70.3%). The results of elemental analysis were asfollows.

Element analysis: calculated: C %=84.05; H %=5.77; found: C %=84.96; H%=5.87.

Reference Example 2

Synthesis of Intermediate of Labeled Compound

The intermediate having the following structure was synthesized.

To anhydrous ether (Et₂O, 30 g) were added NaOCH₃ (3.0 g),4,4′-diacetyl-o-terphenyl (10 mmol) and C₃F₇COOC₂H₅ (20 mmol), themixture was stirred in a sealed container at room temperature for 24 hr.Anhydrous ether was distilled away to give a residue, which was dried invacuo for 30 min. The product was neutralized with 15% sulfuric acid(100 ml), and the precipitate was separated by filtration and washedwell with water. The precipitate was dissolved in ethanol (200 ml) underheating, and the insoluble substance was removed by filtration. Thesolution was concentrated to about 20 ml under reduced pressure. Thissolution was gradually added dropwise to petroleum ether (200 ml) understirring. The mixture was sufficiently stirred and filtered to remove asmall amount of deposited precipitate. The filtrate was evaporated tocompletely remove the organic solvents. The obtained oily substance wasdried in vacuo to give a yellow powder. The product was dried in vaacofor 24 hr. The yield was 460 g (65.0%).

Elemental analysis: calculated, C %=51.00; H %=2.28; found: C %=51.22; H%=2.61

¹H-NMR confirmed that the product was the objective compound.

Reference Example 3

Synthesis of Labeled Compound

The labeled compound having the following structure was synthesized.

To chlorosulfuric acid (3.5 ml) was gradually added β-diketone (theintermediate obtained in Reference example 2, 2 mmol) under stirring atroom temperature. The reaction mixture was stirred at room temperaturefor 7 hr, then carefully added dropwise to ice water (150 ml, outsidecooled with ice water) under stirring. The resultant precipitate wasimmediately centrifuged, washed with cold water (about 5° C.) andcentrifuged twice. The precipitate was suspended in a small amount ofcold water and transferred onto a glass filter, and water was removed bysuction filtration. The resultant chlorosulfonylated β-diketone wasdried in vacuo at room temperature for 48 hr or more. The yield was 77%.

Elemental analysis: calculated: C %=44.76; H %=1.88; found: C %=44.50; H%=1.92.

¹H-NMR confirmed that the product was the objective compound.

EXAMPLE 1

Labeling of p53, Human, Probe (Exon 4 Translated) a Labeled Compound

The labeled compound obtained in Reference Example 3 and p53, Human,Probe (exon 4 translated) manufactured by Oncogene Research Product(Cosmo Bio) were reacted in the following manner to prepare a labeledDNA wherein said labeled compound is bound with p53, Human, Probemediated by a sulfonyl group.

One hundred pmol of p53, Human, Probe (exon 4 translated) was dissolvedin 0.1 mol/l carbonate buffer solution (pH=9.3, 100 μl). To this DNAsolution was gradually added a solution (10 μl) of the labeled compoundhaving a mole number equal to said nucleic acid Waving about 280 aminogroups/molecule) dropwise under stirring at room temperature. Themixture was stirred at room temperature for 1 hr, extracted withphenol/chloroform, and subjected to ethanol precipitation. Theprecipitate was washed with 80% ethanol and dried. The dried precipitatewas re-dissolved in 0.05 mol/l carbonate buffer solution (pH=8.0, 100μl).

The molarity of the labeled compound contained in this solution wascalculated. In addition, the molar absorption coefficient of the labeledcompound at 330 nm was calculated from the absorbance at 330 nm. As aresult, the absorption coefficient was 0.97 mol⁻¹cm⁻¹+l. There was noabsorption by DNA at 330 nm. Assuming that molar absorption coefficientdoes not change during the process of labeling reaction, theconcentration of the label in the labeled DNA solution and the bindingratio of the label and DNA were calculated.

The binding ratio of DNA to the labeled compound obtained by the abovemethod was about 1.

EXAMPLE 2

Hybridization of the Labeled DNA with Genes in a Liver Tissue

A liver tissue excised from human was fixed with 4% paraformaldehyde-PBSat 4° C. overnight, and dehydrated with 70%, 80%, 90/% and 100% ethanol,successively. All water used in this experiment was purified watertreated with diethylpyrocarbonate (DEPC). Further dehydration wasperformed by exchanging the solution in the liver tissue twice with 100%ethanol. Then, the liver tissue was transferred into xylene, heated at60° C. for 2 hr (3 times), and embedded with paraffin.

The section (about 5 μm) of this paraffin-embedded tissue was preparedusing a microtome, placed on a thoroughly washed slide glass, and driedat 37° C. for 6 hr to give a section preparation. The sectionpreparation was further dried with a drier, treated with xylene, 100%ethanol, 90% ethanol, 80% ethanol, 70% ethanol and phosphate buffer (PB,pH 7.4), successively, and treated with proteinase K solution (10 mMTris-HCl (pH 8.0), 1 mM EDTA; 10 μg/ml) for 20 min.

The section preparation was then immersed in 4% paraformaldehyde-PBsolution for 10 min, washed with PB, and treated with 0.2N hydrochloricacid for 10 min. with PB for 1 minute, with 0.1 M triethanolaminehydrochloric acid (pH 8.0) for 1 minute, with 0.1 M triethanolaminehydrochloric acid (pH 8.0) containing 0.25% acetic anhydride for 10 min,and with PB for 1 minute. The section preparation was further treatedwith 70% a ethanol, 80% ethanol, 90% ethanol and 100% ethanol,successively, and air-dried to give an air-dried sample. The air-driedsample was immediately subjected to the following hybridization.

As a hybridization solution, a solution comprising 50% formamide, 10 mMTris-HCl (pH 7.6), 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, 1 MMEDTA, 200 μg/ml tRNA and 1×Denhardt's solution was prepared.

The labeled DNA obtained in the Example was dissolved in thishybridization solution to the concentration of 5 ng/ml. The mixture(about 40 ml) was dropped onto the above air-dried sample. Prior tohybridization, the sample was prehybridized with the hybridizationbuffer without the labeled DNA at 37° C. for 2 hr. Then, the air-driedsample was covered with palm (CAN Co.), and incubated at 37° C. for 16hr in a moisture chamber, in which a paper towel moistened with 50% aformamide solution was set, for hybridization with the labeled DNA.

After hybridization, the parafilm was removed from the slide glass in5×SSC solution (40° C.), and the slide glass having the sectionhybridized with the labeled DNA was heated in 2×SSC, 50% formamide at40° C. for 30 min. The slide glass was washed with TNE solution (aqueoussolution containing Tris-HCl buffer, NaCl, and EDTA), and reacted with 5μg/ml RNase A in TNE solution for 10 min.

The slide glass was then washed with 2×SSC at 40° C. for 20 min (once),and 0.2×SSC at 40° C. for 20 min (twice). Finally, the slide glass wasimmersed in 0.2×SSC solution (pH 8.5) containing 0.1 mM europiumchloride to give a fluorescent complex from the reaction of the labeledDNA with europium chloride. The section on the slide glass was observedwith a fluorescence microscope.

As a result, signals derived from the fluorescent complex of the labeledDNA and europium chloride were detected on the section. Whenhybridization was performed using DNA labeled with fluoresceinisothiocyanate (FITC) in the place of the labeled DNA above as acomparative example, hybridization signals were hardly detected. In thisexperiment, europium chloride was not added to detect the signals.

EXAMPLE 3

Preparation of a Labeled PCR Product

Two oligonucleotides (SEO ID: NO: 1 and SEQ ID: NO: 2), having thenucleotide sequences homologous to the sense and antisense sequence ofhepatitis B virus surface antigen (HBsAg) gene, respectively, weresynthesized by phosphoamidite method using an automatic DNA/RNAsynthesizer. In the final step of the synthesis, a labeled compound wasreacted with the 5′ termini of the elongated oligonucleotides to givetwo kinds of labeled oligonucleotides shown in the following formulas(1) and (2).

wherein Q¹ is oligonucleotide (SEQ ID: NO: 1) in which the amino groupof the nucleotide at the 5′ terminus binds to the sulfonyl group of thelabeled compound.

wherein Q² is oligonucleotide (SEQ ID: NO: 2) in which the amino groupof the nucleotide at the 5′ terminus binds to the sulfonyl group of thelabeled compound.

Using the above two labeled oligonucleotides as a pair of primers andthe nucleic acid fraction extracted with phenol/chloroform from theserum derived from a patient, who was strongly positive against HBsAg,as a template, PCR was carried out under the following conditions.

A reaction mixture consisting of 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5mM magnesium chloride, 0.1% Triton X-100, dNTPs (50 μM each), 0.02 U/μlTaq DNA polymerase, and primers (0.4 pM each) was used foramplification.

After preheating at 95° C. for 2 min, thermal denaturation was performedat 95° C. for 30 sec, annealing was performed at 57° C. for 30 sec, andelongation was performed at 72° C. for 80 sec. These steps were repeated30 cycles to give a 600 bp DNA as a PCR product.

Addition of europium chloride to the DNA product resulted in thegeneration of extremely strong fluorescence.

EXAMPLE 4

Analysis of Liver Tissue Using Labeled PCR Product

A tissue section was prepared from the liver excised from a humaninfected with hepatitis B according to the method described in Example2, except the use of a normal sterilized purified water in the place ofDEPC-treated water, to give an air-dried sample on a slide glass.

A hybridization solution consisting of 50% formamide, 10 mM Tris-HCl (pH7.6), 10% detran sulfate, 600 mM NaCl, 0.25% SDS, 1 mM EDTA, 200 μg/mltRNA and 1×Denhardt's solution,was prepared. Prior to hybridization, theair-dried sample was prehybridized with this hybridization solutionwithout PCR amplification product for 2 hr.

The labeled PCR amplification product obtained in Example 3 wasdissolved in this hybridization solution to the concentration of 70ng/ml in a DNA content. The mixture was preincubated at 85° C. for 10min, and diluted 10fold with the hybridization solution. Thishybridization solution (about 40 μl) was added dropwise onto saidair-dried sample on the slide glass. The slide glass was covered with aparafilm and heated at 95° C. for 2 min on a hotplate.

This slide glass was incubated at 37° C. for 16 hr in a moisturechamber, in which a paper towel moistened with 50% formamide solutionwas set, to allow the air-dried sample to hybridize with the labeled PCRamplification product.

After hybridization, the parafilm was removed from the slide glass in5×SSC solution (40° C.), and the slide glass was heated in 2×SSC and 50%formamide at 40° C. for 30 min. Then, the slide glass was washed withTNE solution, and reacted with 5 μg/ml RNase in TNE solution for 10 min.The slide glass was washed successively with 2×SSC at 40° C. for 20 min(once), and with 0.2×SSC at 40° C. for 20 min (twice). The slide glasswas immersed in 0.2×SSC solution (pH 8.5) containing 0.1 mM europiumchloride, and the tissue section on the slide glass was observed with afluorescence microscope.

As a result, a mosaic staining pattern was detected in liver cells inthe lobulus on the slide glass. As a comparative example, hybridizationwas tried using FITC in the place of the labeled compound mentionedabove. However, the fluorescence of FITC was significantly degraded whenan FITC-labeled PCR product was obtained, so that the subsequenthybridization step could not be performed.

Thus, as an alternative, a DNA product was obtained by amplificationusing unlabeled oligonucleotides having the nucleotide sequencesdepicted in SEQ ID: NO: 1 and SEQ ID: NO: 2 as a pair of primers and anucleic acid fraction derived from an HBsAg strongly positivepatient-derved serum as a template. Said amplification product wasreacted with FITC to give a fluorescence-labeled probe, which wassubjected to hybridization, wherein no europium chloride was added.Hybridization using said FITC-labeled probe gave very weak signals,which showed that the FITC-labeled prove was obviously inferior to thelabeled probe of the present invention.

EXAMPLE 5

Analysis of Human Pancreas Tissue Using Labeled Human Insulin

A labeled human insulin was prepared using a standard human insulin(Sigma) in the same manner as in Example 1.

A pancreas tissue excised from a human was treated in the same manner asin Example 2 to give a section. This section was placed on a slide glassand immersed in 4% formamide solution at room temperature for 10 min. Tothis section was added TBS solution [Tris-NaCl buffer (pH 7.6), 50 μl ]containing 5% skim milk. The section was heated at 37° C. for 2 hr, andwashed with TBS solution (pH 7.6) 3 times.

As a hybridization solution, a solution of 1 mM EDTA and 0.2% BSA in TBSsolution (pH 7.6) was prepared.

The labeled human insulin was dissolved in this hybridization solutionto the concentration of 10 ng/ml, and the mixture (about 40 μl) wasdropped onto the pancreas tissue section. The slide glass was coveredwith parafilm, and incubated at 37° C. for 8 hr in a moisture chamber inwhich a paper towel moistened with 50% formamide solution was set, toallow the section to hybridize with the labeled probe.

After hybridization, the parafilm was removed from the slide glass in5×SSC solution (40° C.), and the slide glass was heated in 2×SSC and 50%formamide at 40° C. for 30 min. Then, the slide glass was washed withTNE solution, and reacted with 5μg/ml RNase in TNE solution for 10 min.The slide glass was washed successively with 2×SSC at 40° C. for 20 min(once), and with 0.2×SSC at 40° C. for 20 min (twice). The slide glasswas immersed in 0.2×SSC solution (pH 8.5) containing 0.1 mM europiumchloride, and the tissue section on the slide glass was observed with afluorescence microscope.

As a result, signals derived from the fluorescent complex of the labeledhuman insulin and europium chloride were detected on the tissue section.

When hybridization was performed using an anti-human insulin receptorantibody (Austral Biologicals (ABI)) labeled with rhodamine in the placeof the labeled human insulin mentioned above as a comparative example,almost the same level of fluorescence image was obtained.

Accordingly, it was concluded that the labeled human insulin of theinvention specifically reacted with a human insulin receptor.

EXAMPLE 6

Analysis of Chromosome Preparation Derived from Peripheral Blood CellUsing Denatured DNA Probe

(Culture of Peripheral Lymphocyte)

Sterilely obtained peripheral blood supplemented with heparin (1 ml) andRPMI1640 medium (GIBCO BRL, 9 ml) supplemented with 15% fetal calf serumwere mixed, and transferred into a culture flask. Phytohemagglutinin(Welcome) was added to the final concentration of 10 g/ml, and theperipheral blood was cultured in a CO₂ incubator with 5% CO₂ atmosphereat 37° C. After 48 hrs of culture, thymidine (Sigma) was added to thefinal concentration of 300 μg/ml, and the culture was continued. At 63hr after the start of the culture, peripheral blood cells comprisinglymphocytes were transferred to a new tube, and centrifuged at 1,200 rpmfor 5 min. The cells were rinsed by adding RPMI1640 medium (10 ml) tothe tube and gently stirring. Said rinsing step was repeated once.RPMI1640 medium supplemented with 15% fetal calf serum (10 ml) and theperipheral blood cells comprising lymphocytes were mixed, and cultured.At 63.5 hr after the start of the culture, bromodeoxyuridine (Sigma) wasadded to the final concentration of 50 ng/ml, the mixture was stirred,and the culture was continued. At 70 hr after the start of the culture,the peripheral blood cells comprising lymphocytes were harvested bycentrifugation at 1,200 rpm.

(Preparation of Chromosome)

To the harvested peripheral blood cells was added 0.075 M KCl (10 ml),and the suspension was stood at room temperature for 30 min. Afterhypotonization, the suspension was centrifuged at 1,200 rpm for 5 min.The supernatant (about 3 ml) and the precipitate were sufficientlystirred using a Pasteur pipette, and gradually dropped into methanol:acetic acid (3:1, carnoy solution, 10 ml). The mixture was sufficientlystirred, stood for 10 min and centrifuged at 1,200 rpm for 5 min. Thesupernatant was removed. To the precipitate was added a fresh carnoysolution (10 ml), and the both were mixed with stirring. This step ofwashing with carnoy solution was repeated twice. The concentration ofthe suspension was adjusted with caroy solution. The suspension wasdropped onto the center of a slide glass, which was followed by steamfixation using a pot containing boiled water.

The fixed sample on the slide glass was dried at 37° C. overnight, whichwas followed by adhesion in a dry heat sterilizer at 65° C. for 4 hr togive a chromosome preparation. This chromosome preparation was stainedwith 2×SSC containing 1 μg/ml fluorescent dye, Hoechst 33258 (Molecularprobe) for 5 min, gently rinsed with 2×SSC and covered with a coverglass, which was followed by standing on a hot plate (75° C.) for 3 min.The preparation was exposed to UV light on the hot plate at a distanceof 1 cm from the preparation with a black light (20 W, Toshiba). Thecover glass on the slide glass was removed and the slide glass wasrinsed twice with distilled water, dried and stored at −20° C. with thechromosome preparation carried thereon.

(Preparation of Labeled dUTP)

A labeled dUTP was prepared using dUTP (deoxy UTP, Toyo Boseki) in thesame manner as in Example 1.

This labeled dUTP and KRAS ONCOGENE (Lab Logics, large probe) weresubjected to nick translation method to give a labeled nucleic acid.

The nick translation followed the protocol using a nick translation kitmanufactured by Boehringer. The labeled dUTP was used at a finalconcentration of 0.05 mM.

After the nick translation, 4 M ammonium acetate (2.5 μl), 10 mg/mlsalmon sperm DNA (Sigma, 2.0 μl), 10 mg/ml E. coli tRNA (Sigma, 2.0 μl)and special grade ethanol (75 μl) were added to the nick translationreaction mixture (20 μl), admixed well, stored at −80° C. for 1 hr, andcentrifged at 15,000 rpm to give precipitate, which was stirred inspecial grade formamide to dissolution. (hybridization)

To the labeled nucleic acid (5 μl) prepared according to theabove-mentioned method was added 10 mg/ml Cot-1 DNA (manufactured byVysis, 5 μl) and incubated in a heat block at 70° C. for 10 min todenature the labeled probe to give a denatured DNA probe, which wasquickly cooled in ice water.

Then, the above-mentioned slide glass carrying the chromosomepreparation was immersed in a coupling jar filled with 70° C. formamide(2×SSC) at 70° C. to denature the sample with heat. The sample wasimmediately moved into 70% ethanol at −20° C., rapidly cooled for 2 min,dehydrated with 100% ethanol and dried. The above-mentioned denaturedDNA probe was mixed in a solution having a final concentration of 50%formamide and 10% dextran sulfate (in 2×SSC) and placed on theabove-mentioned chromosome preparation. The denatured DNA probe solutionwas uniformly spread thereon using a parafilm strip to prevent inclusionof air foams. The chromosome sample on the slide glass and the denaturedDNA probe were hybridized in a sealed moistened chamber containing afilter paper impregnated with 2×SSC on the bottom therein at 37° C. for18 hr.

The parafilm was stripped off the slide glass and the slide glass wasimmersed in a coupling jar filled with 50% formamide (in 2×SSC) at 37°C. and rinsed for 15 min. This slide glass was stood still in 2×SSC(room temperature) for 1 min and then stood still for 15 min in 1×SSC(room temperature) and 5 min in 4×SSC (room temperature). Then, 2×SSC(pH 8.5) containing 0.1 mM europium chloride was dropped, on a slideglass and the tissue section strip on the slide glass was observed witha fluorescence microscope.

As a result, the 11th chromosome was found to have fluorescence, whichcoincided with the localization of nucleic acid containing KRASONCOGENE, thereby con fining possible specific detection.

EXAMPLE 7

(Preparation of Fluorescence-labeled Streptoavidin)

Recombinant streptoavidin (24 mg, Boehringer Mannheim) was dissolved in100 mM carbonate buffer (pH 9.3, 2 ml) and dialyzed against the samebuffer. From the absorbance of dialysis solution at 280 nm, it wasconfirmed to have a 3.4 mg/ml protein concentration. To the entireamount of 2 ml thereof was dropwise added an anhydrous DMF solution (0.4ml) containing the labeled compound (7.4 mg) of Reference example 3 andthe mixture was stirred at 25° C. for 1 hr. After stirring, the reactionmixture was eluted with 50 mM ammonium carbonate using Sephadex G-50column (about 30 ml bed) to separate a non-reacted labeled compound.From the absorption coefficient 3.41×10⁴ (cm⁻¹M⁻¹) at 330 nm of theprotein fraction and the molecular weight of recombinant streptoavidinof about 52,000, the cross-linked labeled compound was calculated to beabout 20 molecules per 1 molecule of streptoavidin. Sodium azide wasadded to the protein fraction to the concentration of 0.1%, adjusted topH 6.5 with IN HCl and stored at 4° C.

(Preparation of DNA Probe Using Nick Translation Kit)

Using the KRAS ONCOGENE (Lab Logics, large probe) used in Example 6 andbiotin-21-dUTP nick translation kit (Clontech), and following theprotocol attached to the kit, biotin-21-dUTP was incorporated into thenucleic acid KRAS ONCOGENE to give a biotinylated KRAS ONCOGENE DNAprobe. The biotin-21dUTP has the following structure including a linkagegroup.

wherein R has the formula

To the reaction mixture (20μl) after nick translation reaction wereadded 4 M ammonium acetate (2.5 μl), 10 mg/ml salmon sperm DNA (Sigma,2.0 μl), 10 mg/ml E. coli TRNA (Sigama, 2.0 μl) and special gradeethanol (75 μl) and admixed well. After storing at −80° C. for 1 hr, itwas centrifuged at 15,000 rpm and the precipitate was thoroughly stirredin special grade formamide to dissolution. (hybridization)

Completely in the same manner as in Example 6, a denatured DNA probe wasprepared and hybridized with a chromosome preparation.

After hybridization, a parafilm was stripped off the slide glass and theslide glass was immersed in a coupling Jar filled with 50% formamide (in2×SSC ) at 37° C. and rinsed for 15 min. This slide glass was stoodstill in 2×SSC (room temperature) for 1 min and then stood still for 15min in 1×SSC (room temperature) and 5 min in 4×SSC (room temperature).The labeled streptoavidin prepared above was diluted with 2×SSC to theconcentration of 0.02 mg/ml. The slide glass was left standing still inthe solution at room temperature for 15 min. Using 1×SSC (roomtemperature), the slide glass was left standing still for 5 min, whichstep was repeated three times. Then, 2×SSC (pH 8.5) containing 0.1 mMeuropium chloride was dropped on a slide glass and the tissue sectionstrip on the slide glass was observed with a fluorescence microscope.

As a result, the 11th chromosome was found to have fluorescence likeExample 6, but apparently had stronger fluorescence. This fluorescenceintensity was considered to reflect the high incorporation rate ofbiotinylated-21-UTP into the nucleic acid and the great number of thelabeled compounds bonded to streptoavidin. The synchronized localizationwith KRAS ONCOGENE was confirmed and the method was concluded to be aspecific detection method.

This application is based on patent application Nos. 119768/1998 and184852/1998 filed in Japan, the contents of which are herebyincorporated by reference.

2 1 27 DNA Artificial Sequence Oligonucleotide designed to act as senseprimer for amplifying HBsAg gene. 1 catggagaac atcacacatc aggattc 27 224 DNA Artificial Sequence Oligonucleotide designed to act as antisenseprimer for amplifying HBsAg gene. 2 aatgtatacc cagagacaaa acaa 24

What is claimed is:
 1. A labeled nucleic acid probe comprising a labelsubstance of the formula (I):

wherein A¹ is an aromatic group, R¹ is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, or a label substanceof the following formula (II):

wherein A² and A³ are the same or different and each is an aromaticgroup, R² and R³ are the same or different and each is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, bonded to a nucleicacid probe via a crossing-linking group.
 2. The labeled nucleic acidprobe of claim 1, wherein the label substance has the formula (III):

wherein n is an integer of 1-6.
 3. The labeled nucleic acid probe ofclaim 1, wherein the cross-linking group is a sulfonyl group or acarbonyl group.
 4. The labeled nucleic acid probe of claim 1, whereinthe label substance is bonded to the nucleic acid probe via aconjugating group.
 5. The labeled nucleic acid probe of claim 4, whereinthe conjugating group is a divalent aliphatic hydrocarbon having 5 to 25carbon atoms and optionally having 7 or less amide bonds betweencarbons.
 6. The labeled nucleic acid probe of claim 5, wherein theconjugating group has the formula (IV):—CH═CHCO—NH_(b)CH₂_(a)CO—NH_(b)CH₂_(a)CO—NH_(b)  (IV) whereina is an integer of 0-6 and b is 0 or
 1. 7. The labeled nucleic acidprobe of claim 4, wherein the conjugating group comprises anaffinity-bound biotin and an avidin.
 8. The labeled nucleic acid probeof claim 7, wherein the biotin is amide-bound with a divalent aliphatichydrocarbon having 5 to 25 carbon atoms and optionally having 7 or lessamide bonds between carbons.
 9. The labeled nucleic acid probe of claim8, wherein the aliphatic hydrocarbon is—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂— which is bonded toa probe.
 10. A fluorescent complex comprising the labeled nucleic acidprobe of claim 1 and a heavy metal ion.
 11. The fluorescent complex ofclaim 10, wherein the heavy metal ion is a lanthanoide metal ion orradium ion.
 12. The fluorescent complex of claim 11, wherein thelanthanoide metal ion is a member selected from the group consisting ofions of europium, samarium, terbium, dysprosium and a mixture thereof.13. A reagent for analyzing a nucleic acid, comprising the labelednucleic acid probe of claim
 1. 14. The reagent for analyzing claim 13,comprising a heavy metal ion.
 15. The reagent for analyzing of claim 14,wherein the heavy metal ion is a lanthanoide metal ion or radium ion.16. A labeled nucleotide comprising a label substance of the formula(I):

wherein A¹ is an aromatic group, R¹ is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, which is bonded to anucleotide via a cross-linking group.
 17. The labeled nucleotide ofclaim 16, wherein the nucleotide is dUTP.
 18. The labeled nucleotide ofclaim 16, wherein the cross-linking group is a sulfonyl group orcarbonyl group.
 19. The labeled nucleotide of claim 16, wherein thelabel substance is bonded to a nucleotide via a conjugating group. 20.The labeled nucleotide of claim 19, wherein the conjugating group is adivalent aliphatic hydrocarbon having 5 to 25 carbon atoms andoptionally having 7 or less amide bonds between carbons.
 21. The labelednucleotide of claim 20, wherein the conjugating group has the formula(IV): —CH═CHCO—NH_(b)CH₂_(a)CO—NH_(b)CH₂_(a)CO—NH_(b)  (IV)wherein a is an integer of 0-6 and b is 0 or
 1. 22. The labelednucleotide of claim 19, wherein the conjugating group comprises anaffinity-bound biotin and avidin.
 23. The labeled nucleotide of claim22, wherein the biotin is amide-bound with a divalent aliphatichydrocarbon having 5 to 25 carbon atoms and optionally having 7 or lessamide bonds between carbons.
 24. The labeled nucleotide of claim 22,wherein the aliphatic hydrocarbon is—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂— which is bonded toa probe.
 25. A fluorescent complex comprising the labeled nucleotide ofclaim 16 and a heavy metal ion.
 26. The fluorescent complex of claim 25,wherein the heavy metal ion is a lanthanoide metal ion or radium ion.27. The fluorescent complex of claim 26, wherein the lanthanoide metalion is a member selected from the group consisting of ions of europium,samarium, terbium, dysprosium and a mixture thereof.
 28. A method forproducing a labeled nucleic acid probe comprising reacting the labelednucleotide of claim 16, dNTPs and a single strand DNA in the presence ofa primer and a DNA polymerase.
 29. A labeled nucleic acid probe obtainedby the production method of claim
 28. 30. A method for producing alabeled nucleic acid probe comprising reacting the labeled nucleotide ofclaim 16, dNTPs and a double stranded DNA in the presence of5′-exonuclease, DNase and a DNA polymerase.
 31. A reagent for analyzingnucleic acid comprising a label substance of the formula (I):

wherein A¹ is an aromatic group, R¹ is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, or a label substanceof the following formula (III):

wherein A² and A³ are the same or different and each is an aromaticgroup, R² and R³ are the same or different and each is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, to which avidin iscovalently bonded via a cross-linking group, and a nucleotide to whichbiotin is bonded via a linkage group.
 32. The reagent for analysis ofnucleic acid of claim 31, comprising a dNTP primer, a DNA polymerase anda heavy metal.
 33. The reagent for analysis of nucleic acid of claim 31,comprising dNTPs, 5′-exonuclease, DNase, a DNA polymerase and a heavymetal.
 34. The reagent for analysis of nucleic acid of claim 31, whereinthe cross-linking group is a sulfonyl group or carbonyl group.
 35. Thereagent for analysis of nucleic acid of claim 31, wherein the nucleotideis dUTP.